WO2020107566A1 - 一种小分子蛋白质及其应用 - Google Patents

一种小分子蛋白质及其应用 Download PDF

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WO2020107566A1
WO2020107566A1 PCT/CN2018/122067 CN2018122067W WO2020107566A1 WO 2020107566 A1 WO2020107566 A1 WO 2020107566A1 CN 2018122067 W CN2018122067 W CN 2018122067W WO 2020107566 A1 WO2020107566 A1 WO 2020107566A1
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small molecule
plasmid
protein
molecule protein
gem
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李翔
杨海洋
钱政江
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深圳先进技术研究院
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/04—Chelating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This application relates to the field of biotechnology, in particular to a small molecule protein and its application.
  • Magnetic resonance imaging is an imaging technique that uses signals generated by resonance of atomic nuclei in a strong magnetic field to reconstruct images.
  • MRI uses radio frequency pulses to excite nuclei containing spins that are not zero in the magnetic field. After the radio frequency pulses stop, the nuclei are imaged.
  • the induction coil is used to collect signals, and the images are reconstructed according to certain mathematical methods. That is, the organism is placed in a special magnetic field, and radio frequency pulses are used to excite the hydrogen nuclei in the organism, causing the hydrogen nuclei to resonate and absorb energy.
  • the hydrogen nucleus After stopping the radio frequency pulse, the hydrogen nucleus emits a radio signal at a specific frequency and releases the absorbed energy, which is recorded by a receiver outside the body and processed by an electronic computer to obtain an image.
  • MRI technology is currently widely used in scientific research and medical detection imaging. It has the safety of no radiation damage and can be scanned in any orientation. However, it is clinically found that the imaging times of different tissues or tumor tissues overlap with each other, which makes diagnosis difficult. Therefore, people began to study contrast agents to enhance signal contrast and improve image resolution. This process is mainly to change the local imaging characteristics of the tissue by injecting contrast agent and improve the imaging contrast.
  • the commonly used contrast agent is a kind of chemically synthesized substance whose density is higher than that of living tissue.
  • the contrast agent itself does not generate a signal.
  • paramagnetic positive contrast agents include Gd-DTPA, Mn-DPDP, etc., which mainly shortens T1 and shows a high signal on T1-weighted images.
  • Superparamagnetic substances such as superparamagnetic iron oxide particles, etc., whose main function is to shorten T2 and show a low signal on the T2 weighted image.
  • CN103432599A discloses a preparation method of nanometer trimanganese tetraoxide nuclear magnetic resonance contrast agent.
  • the method uses pulse laser to ablate solid target material in liquid to synthesize micro-nano material. Its operation is simple and there is no other chemical impurities.
  • CN103191446A provides a method for preparing a nuclear magnetic resonance contrast agent. The iron salt is reduced at a high temperature with reducing sugar to obtain Fe 3 O 4.
  • CN102397564A discloses a polypeptide-modified tumor targeting diagnostic nuclear magnetic resonance contrast agent and a preparation method thereof, which uses a polymer material, polyethylene glycol, a polypeptide, a bifunctional ligand and gadolinium trichloride, and uses the polypeptide as a targeting head group
  • the dendritic polymer material is a basic polymer carrier, and a small molecule contrast agent is connected to the surface to make a MRI contrast agent for tumor targeted diagnosis.
  • contrast agents have strong biological toxicity, such as gadolinium (Gadolinium) chelates
  • this type of contrast agent is one of the most commonly used contrast agents in recent decades, but such metal chelates
  • Contrast agents will accumulate in large amounts in the brain, kidneys, bones, and other tissues, and have a strong biological toxicity to the body;
  • the contrast agent is metabolized faster, not suitable for long-term scientific research or clinical medical observation; existing contrast agents
  • the metabolic rate in the organism is relatively fast, and most of them will be reduced to an effective dose after 48-72h. Therefore, for long-term continuous or multiple observation imaging, the existing contrast agents are not enough.
  • the research provides a new type of MRI contrast agent with tissue cell specificity, strong penetrating power, and no biological toxicity, which has broad application prospects and huge market value.
  • this application provides a small molecule protein and its application.
  • the present application provides a small molecule protein, and the amino acid sequence of the small molecule protein is shown in SEQ ID NO.1.
  • the SEQ ID NO.1 is as follows:
  • the affinity metal ions of the small molecule protein include divalent manganese ions.
  • nucleotide sequence of the small molecule protein is shown in SEQ ID NO. 2;
  • sequence of SEQ ID NO. 2 is as follows:
  • the small molecule protein is a protein less than 15KD.
  • GEM GEM
  • the present application provides a plasmid that includes a nucleotide sequence encoding the small molecule protein of the first aspect.
  • the present application provides a lentivirus, which is obtained by co-transfection and packaging of the plasmid and the auxiliary plasmid of the second aspect.
  • the present application provides a use of the small molecule protein described in the first aspect, the plasmid described in the second aspect, or the lentivirus described in the third aspect for the preparation of a contrast agent for magnetic resonance imaging.
  • the present application provides a use of the small molecule protein described in the first aspect, the plasmid described in the second aspect, or the lentivirus described in the third aspect for preparing a nerve tracer.
  • the present application provides a use of the small molecule protein according to the first aspect, the plasmid according to the second aspect or the lentivirus according to the third aspect for preparing a marker for detecting gene expression
  • the applicant has thoroughly researched the development status of clinical MRI contrast agents, extensively summarized the advantages and disadvantages of the existing technology, and used genetic engineering as a means to discover a new small molecule protein with an amino acid sequence such as SEQ ID NO.1
  • the small molecule protein can be used as a specially modified gene-encoded MRI contrast agent.
  • This gene sequence is expressed in vivo by lentivirus and other methods, and the expressed protein can bind in vivo Manganese ions, because manganese is a paramagnetic substance, can cause a significant reduction in T1 relaxation time during nuclear magnetic imaging, and high signals in the relevant parts during imaging.
  • the small molecule protein solves many problems such as poor tissue penetration, no cell/tissue selectivity, strong biological toxicity (metal contrast agent) and the like commonly used in MRI contrast agents; High-resolution, real-time, in vivo detection of gene expression, cell differentiation, individual development process, etc., has broad application prospects.
  • the present application provides a method for preparing a small molecule protein as described in the first aspect, including the following steps:
  • the protein expression in step (2) includes lentiviral packaging of the GEM plasmid and infection of cells or tissue injection.
  • the protein expression vector in step (2) includes any one or a combination of at least two of Hela cells, rat brain tissue or rhesus monkey brain tissue.
  • tissue-specific expressions (or non-specific expressions) can be performed according to different experimental or clinical needs.
  • This application takes lentivirus-mediated expression in Hela cells and rat brain tissue as an example, but it is not limited to this; lentivirus packaging, viral cell infection and intracerebral microinjection methods are commonly used general techniques and will not be repeated here. .
  • the present application provides a method for preparing a small molecule protein according to the first aspect, which specifically includes the following steps:
  • step (2) Pack the plasmid constructed in step (1) with lentivirus and infect cells or inject tissues for protein expression.
  • step (1) in order to more easily detect the expression of GEM, a 3xFlag tag sequence is inserted at its 3'end. Therefore, the final insertion sequence of pUltra-Smurf is shown in SEQ ID NO.3:
  • the first is to inject nano-scale metal particles into biological tissues, and the other expresses transferrin gene to accumulate endogenous iron. Due to the different distribution of iron, MRI produces T2-weighted imaging
  • the contrast signal of these two schemes has the following shortcomings: First, the nano-metallic particles have poor tissue penetration and no tissue-specific selectivity; second, although the expression of transferrin has tissue-specific selectivity and Good tissue selectivity, but iron belongs to T2 contrast agent, and the principle of T2 contrast agent is to reduce the signal-to-noise ratio, that is, it is darker on the dark basis, so the resolution is poor and the signal is relatively insignificant.
  • the small molecule protein provided by this application is a gene-mediated T1 type positive contrast agent.
  • different promoters can be selected for specific expression according to different tissues/cells, so it has a strong Tissue/cell specific selectivity solves the problems of poor tissue penetration and no cell/tissue selectivity of current commonly used MRI contrast agents;
  • T1 Contrast agent the principle of T1 type contrast agent is to improve the signal-to-noise ratio, so the imaging resolution is higher and the effect is better than T2 type contrast agent;
  • this application can artificially add different gene expression switches according to different clinical or scientific research needs ”Components, select the expression of different time periods, slow metabolism, suitable for long-term clinical observation and scientific research, so it solves the problem of strong biological toxicity (metal contrast agent), and can detect gene expression in high resolution, real time, in vivo , Individual development process of cell differentiation, etc.
  • Figure 1 is a graph of the expression of GEM in the cell of this application, where Figure 1(A) is the Western blot result of HeLa cells transfected with GEM; Figure 1(B) is a diagram of the expression of GEM in the rat brain;
  • Figure 2(A) is an MRI scan of Hela cells after expressing GEM
  • Figure 2(B) is a histogram of cell MRI signal strength
  • Figure 3(A) is a 3T MRI T1 coronal scan of GEM expressed in rat brain tissue
  • Figure 3(B) is a 3T MRI T1 horizontal scan of GEM expression in rat brain tissue
  • Figure 3(C) is the corresponding map of virus injection and MRI results
  • Figure 4(A) is the original technology T2 weighted image
  • Fig. 4(B) is a T1 weighted imaging effect diagram of the present application.
  • Figure 1(A)- Figure 1(B) The virus expression effect is shown in Figure 1(A)- Figure 1(B).
  • Figure 1(A) is the Western blot test result after HeLa cells are transfected with GEM.
  • Figure 1(B) is the virus expression in rat brain.
  • 1(A) and FIG. 1(B) show that the protein expression is successful.
  • Intra-brain virus microinjection In this example, the rat striatum brain area was selected for verification (the striatum is a relatively large and uniform nucleus, which is relatively isotropic, and other brain areas can also be used as experimental brain areas) ; After anesthetized by intraperitoneal injection of chloral hydrate in adult rats, fix the rat prone on the stereotactic position, cut the scalp in the middle and separate the periosteum, according to the position of the striatum nucleus in the brain map of the rat M/L 3.0, D/V 5.2) Use a skull drill to make a small hole in the skull, and slowly inject 2000nL of lentivirus into the striatum brain area with a 10uL microinjection needle at a sampling rate of 100nL/min. After the injection is completed, stop Needle 5min, slowly remove the needle.
  • the rat brain tissue after expression in Example 3 was subjected to MRI scan detection, and 5-6 weeks after virus injection, the rats were anesthetized and then scanned by MRI (uMR790, Shanghai United Imaging Healthcare), using a stereo pixel resolution of 0.25*0.25*1.5mm three-dimensional magnetization preparatory gradient echo sequence, collect T1-weighted images of the coronal and horizontal planes of the whole brain of the rat, and scan each side for 4-6 minutes; the results are shown in Figure 3(A)- Figure 3( C), because GEM can express manganese ions after being expressed in this application, and manganese ions are paramagnetic substances, so T1 weighted images were collected during MRI scan.
  • Figure 3(A) shows the results of the coronal plane scan of the rat brain
  • Figure 3( B) is the result of a horizontal scan of the rat brain
  • Figure 3(C) is a map of virus injection and MRI results.
  • the lentivirus uses a highly expressed ubiquitin promoter Ub, where Amcyan is a fluorescent protein tag used to detect viruses
  • the expression on the left of the virus in the figure is a control; the results show that the GEM of this application can produce a high signal on the T1 weighted image, and the signal of the MRI and the signal expressed by the virus are coincident.
  • the small molecule protein provided by this application is a specially modified gene-encoded MRI contrast agent. After being expressed in vivo, it can cause a significant reduction in T1 relaxation time during MRI, and the relevant parts are high in imaging.
  • the signal solves many problems such as poor tissue penetration, no cell/tissue selectivity, strong biological toxicity (metal contrast agent), and the like, which can detect gene expression in high resolution, real time, in vivo, The process of cell differentiation and individual development has broad application prospects and huge market value.

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Abstract

提供一种小分子蛋白质及其应用，所述小分子蛋白质的氨基酸序列如SEQ ID NO.1所示；所述小分子蛋白为特殊改造的基因编码的核磁共振成像造影剂；本申请提供的基因序列在生物体内表达后，在核磁成像时能造成T1弛像时间的明显缩短，成像时相关部位高信号，解决了目前常用核磁共振成像造影剂的组织穿透性差、没有细胞/组织选择性、生物毒性强(金属造影剂)等众多问题，能够高分辨率、实时、在体地检测基因表达、细胞分化个体发育过程等，具有广阔的应用前景和巨大的市场价值。

Description

一种小分子蛋白质及其应用 技术领域
本申请涉及生物技术领域，尤其涉及一种小分子蛋白质及其应用。
背景技术
磁共振成像(MRI，Magnetic Resonance imaging)是利用原子核在强磁场内发生共振产生的信号经图像重建的一种成像技术。MRI利用射频脉冲对置于磁场中含有自旋不为零的原子核进行激发，射频脉冲停止后，原子核进行成像，在其成像过程中用感应线圈采集信号，按照一定的数学方法重建形成图像。即将生物体置于特殊的磁场中，用无线电射频脉冲激发生物体内氢原子核，引起氢原子核共振，并吸收能量。在停止射频脉冲后，氢原子核按特定频率发出射电信号，并将吸收的能量释放出来，被体外的接收器收录，经电子计算机处理获得图像。MRI技术目前普遍应用于科学研究以及医学检测成像中，具有无辐射损伤的安全性，可任意方位断层扫描等，然而临床上发现不同组织或肿瘤组织的成像时间相互重叠，导致诊断困难。因此人们开始研究造影剂，增强信号对比、提高图像分辨率。这一过程主要是通过注射造影剂来改变组织局部成像特性，提高成像对比度。
目前常用造影剂是一类化学合成的其密度高于活体组织的物质，造影剂本身不产生信号，通过改变生物体内部组织中水质子的成像效率，与周围组织形成对比，从而达到造影的目的。其中，顺磁性阳性造影剂包括Gd-DTPA、Mn-DPDP等，起作用主要使T1缩短，在T1加权图像上呈高信号。超顺磁性物质，例如超顺磁性氧化铁颗粒等，其主要作用是使T2缩短，在T2加权像上呈低信号。
CN103432599A公开了一种纳米四氧化三锰核磁共振造影剂的制备方法，该方法通过脉冲激光在液体中烧蚀固体靶材合成微纳材料，其操作简单，没有其他化学杂质，制备的纳米四氧化三锰核磁共振造影剂的磁豫率高达8.26mM-1s-1，比商业用的Gd-DTPA的值(r1＝4.11mM-1s-1)高出一倍，显示出了良好的体内和体外成像效果。CN103191446A提供了一种核磁共振造影剂的制备方法，用还原性糖将铁盐在高温下还原后得到Fe 3O 4，处理后进行表面修饰，标记上抗体后，注射进大鼠体内一段时间后，即可进行MRI扫描成像。CN102397564A公开了一种多肽修饰的肿瘤靶向诊断核磁共振造影剂及其制备方法，采用高分子材料、聚乙二醇、多肽、双功能配体和三氯化钆，以多肽为靶向头基，树枝状高分子材料为基础高分子载体，表面连接小分子造影剂，制成肿瘤靶向诊断核磁共振造影剂。
但总体来说这几类造影剂的院里都是将金属复合物打入生物体内，其缺点十分明显：一、不具有细胞/组织特异选择性；目前绝大多数造影剂都为外源金属复合物，因此其并不具有组织或者细胞特异性。而当想要在体实时研究某一类基因的表达，或者某一类细胞/组织分化发育过程时，现有造影剂无法完成这一目的；二、穿透性差，具有生物毒性。现有造影剂由于大多为外源金属复合物，因此其对于某些组织或者肿瘤穿透性较差，因此当遇到这一类组织或者肿瘤时，造影剂的效果会有很大程度上的减弱。另外目前常用的许多造影剂都具有很强的生物毒性，例如钆(Gadolinium)类螯合物，这一类造影剂为近几十年来最常用的造影剂之一，但是这类金属螯合物造影剂会在大脑、肾脏、骨头等组织中大量累积，并对机体产生很强的生物毒性；三、造影剂代谢较快，不适合长时程的科研研究或者临床医学观察；现有造影剂在生物体内的代谢速率较快，绝大多数都会在48-72h后降低至有效剂量一下，因此对于长时程连续或者 多次的观察成像，现有造影剂并不能满足。
综上所述，研究提供一种具有组织细胞特异性、穿透力强、无生物毒性的新型核磁共振成像造影剂，具有广阔的应用前景和巨大的市场价值。
发明内容
针对现有技术的不足及实际的需求，本申请提供一种小分子蛋白质及其应用，所述
为达此目的，本申请采用以下技术方案：
第一方面，本申请提供一种小分子蛋白质，所述小分子蛋白质的氨基酸序列如SEQ ID NO.1所示。
所述SEQ ID NO.1如下所示：
Figure PCTCN2018122067-appb-000001
优选地，所述小分子蛋白质的亲和金属离子包括2价锰离子。
优选地，所述小分子蛋白质的核苷酸序列如SEQ ID NO.2所示；
所述SEQ ID NO.2的序列如下：
Figure PCTCN2018122067-appb-000002
Figure PCTCN2018122067-appb-000003
其中，所述小分子蛋白质为小于15KD的蛋白质。
本申请中，申请人发现一种新型小分子蛋白质，命名为GEM，所述小分子蛋白质特异性结合2价锰离子，能够开发基于结合2价锰离子的应用。
第二方面，本申请提供一种质粒，所述质粒包括编码第一方面所述的小分子蛋白质的核苷酸序列。
第三方面，本申请提供一种慢病毒，所述慢病毒通过第二方面所述的质粒和辅助质粒共转染包装得到。
第四方面，本申请提供一种如第一方面所述的小分子蛋白质、第二方面所述的质粒或第三方面所述的慢病毒用于制备核磁共振成像造影剂的用途。
第五方面，本申请提供一种如第一方面所述的小分子蛋白质、第二方面所述的质粒或第三方面所述的慢病毒用于制备神经示踪剂的用途。
第六方面，本申请提供一种如第一方面所述的小分子蛋白质、第二方面所述的质粒或第三方面所述的慢病毒用于制备检测基因表达的标记物的用途
本申请中，申请人通过深入研究临床核磁成像造影剂的发展现状，广泛总结现有技术的优缺点，以基因工程为手段，发现了一种新型小分子蛋白，氨基酸序列如SEQ ID NO.1所示，所述小分子蛋白质能够作为一种经特殊改造的基因编码的核磁共振成像造影剂，此基因序列在经慢病毒等方式介导在生物体内表达，所表达的蛋白在生物体内能结合锰离子，由于锰是一种带有顺磁性的物质，因此在核磁成像时能造成T1弛像时间的明显缩短，成像时相关部高信号。所述小分子蛋白质作为一种T1类型的阳性造影剂，解决了目前常用核磁共振成像造影剂的组织穿透性差、没有细胞/组织选择性、生物毒性强(金属造影剂)等众多问题；能够高分辨率、实时、在体的检测基因表达、细胞分化个体发育 过程等，具有广阔的应用前景。
第七方面，本申请提供一种如第一方面所述的小分子蛋白质的制备方法，包括如下步骤：
(1)以GEM蛋白的核苷酸序列为基础，进行酶切连接，构建GEM质粒；
(2)将步骤(1)构建的质粒进行蛋白表达。
本申请中的质粒构建过程，可根据不同需要选择不同载体进行构建。
优选地，步骤(2)所述蛋白表达的方式包括将GEM质粒进行慢病毒包装并感染细胞或注射组织。
优选地，步骤(2)所述蛋白表达的载体包括Hela细胞、大鼠脑组织或恒河猴脑组织中的任意一种或至少两种的组合。
本申请的蛋白表达过程，可根据不同实验或临床需求，进行不同组织特异性表达(或者非特异性表达)。
本申请以慢病毒介导分别在Hela细胞和大鼠脑组织的表达为例，但并不局限于此；慢病毒包装、病毒细胞感染及脑内微量注射方法为常用通用技术在此不做赘述。
作为优选技术方案，本申请提供一种如第一方面所述的小分子蛋白的质制备方法，具体包括如下步骤：
(1)以氨基酸序列如SEQ ID NO.2所示GEM蛋白的核苷酸序列为基础，在其3’端和5’端以BamHI和EcoRI两种酶插入pUltra-Smurf载体，进行酶切连接，构建GEM质粒；
(2)将步骤(1)构建的质粒进行慢病毒包装并感染细胞或注射组织，进行蛋白表达。
步骤(1)中，为了更为方便的检测GEM的表达，在其3’端插入一段3xFlag  tag序列，因此最终插入pUltra-Smurf的序列如SEQ ID NO.3所示：
SEQ ID NO.3：
Figure PCTCN2018122067-appb-000004
目前现有核磁成像造影剂的技术有两种，第一是向生物组织注入纳米级金属颗粒，另一种表达转铁蛋白基因，累积内源铁，由于铁的不同分布从而产生MRI T2加权成像的对比信号；而这两种方案目前都有以下几点缺点：首先，纳米金属颗粒的组织穿透性较差，并且没有组织特异选择性；其次，表达转铁蛋白虽然具有组织特异选择性以及较好的组织选择性，但是铁类属于T2造影剂，而T2造影剂的原理是降低信噪比，即在暗的基础上边的更暗，因此分辨率较差，信号相对不明显。
与现有技术相比，本申请具有如下有益效果：
(1)本申请提供的小分子蛋白质作为一种基因编码介导的T1类型的阳性造影剂，在使用过程中可以根据不同组织/细胞选择不同的启动子进行特异性表达，因此具有很强的组织/细胞特意选择性，解决了目前常用核磁共振成像造影 剂的组织穿透性差、没有细胞/组织选择性的问题；
(2)本申请所采用的基因序源自于对人人体无害的微生物，并且本申请已经在啮齿类、非人灵长类动物中进行验证，并未发现其生物毒性；本申请属于T1类造影剂，T1类型的造影剂原理为提高信噪比，因此成像分辨率较高，较T2类造影剂效果更好；本申请可根据不同临床或科研需要人为加上不同的基因表达“开关”元件，选择不同时间段的表达，代谢慢，适合长时程临床观察和科学研究，因此解决了生物毒性强(金属造影剂)的问题，能够高分辨率、实时、在体的检测基因表达、细胞分化个体发育过程等。
附图说明
图1为本申请的GEM在细胞内的表达情况图，其中，图1(A)为HeLa细胞转染GEM后的Western blot结果；图1(B)为GEM在大鼠脑中表达情况图；
图2(A)为Hela细胞表达GEM后的MRI扫描图；
图2(B)为细胞MRI信号强度柱状图；
图3(A)为GEM在大鼠脑组织表达的3T MRI T1冠状面扫描图；
图3(B)为GEM在大鼠脑组织表达的3T MRI T1水平面扫描图；
图3(C)为病毒注射与MRI结果对应图；
图4(A)为原有技术T2加权像；
图4(B)为本申请T1加权成像效果图。
具体实施方式
为更进一步阐述本申请所采取的技术手段及其效果，以下结合附图并通过具体实施方式来进一步说明本申请的技术方案，但本申请并非局限在实施例范围内。
实施例1质粒构建
本申请以pUltra-Smurf载体为例，其中GEM原始序列SEQ ID NO.2如下所示：
Figure PCTCN2018122067-appb-000005
分别在其3’端和5’端以BamHI和EcoRI两种酶插入pUltra-Smurf载体，同时为了更为方便的检测GEM的表达，在其3’端插入一段3xFlag tag序列，因此最终插入pUltra-Smurf的序列为SEQ ID NO.3：
Figure PCTCN2018122067-appb-000006
Figure PCTCN2018122067-appb-000007
实施例2 GEM在细胞内的表达
以实施例1得到的质粒与包装质粒进行包装慢病毒，感染Hela细胞；
病毒表达效果见图1(A)-图1(B)，图1(A)为HeLa细胞转染GEM后的Western blot检测结果，图1(B)为大鼠脑内病毒表达情况，由图1(A)和图1(B)可知，蛋白表达成功。
细胞内表达后进行MRI检测，结果如图2(A)和图2(B)所示；
由图2(A)和图2(B)所示，对细胞进行MRI T1加权像扫描，GEM在Hela细胞内表达成功，且MRI信号强度明显高于对照组。
实施例3 GEM在组织内的表达
将包装好的慢病毒进行大鼠脑内病毒微量注射，步骤如下：
脑内病毒微量注射：本实施例选择大鼠纹状体脑区进行验证(纹状体这一核团相对比较大，比较均一，各向同性比较好，其他脑区也可以作为实验脑区)；成年大鼠经水合氯醛腹腔注射麻醉后，将大鼠俯卧固定于立体定位上，头皮正中切开并分离骨膜，根据大鼠脑图谱中纹状体核团的位置(A/P 0.6，M/L 3.0，D/V 5.2)用颅钻在颅骨开一小孔，用10uL微量注射针向纹状体脑区缓慢注射2000nL的慢病毒，进样速度100nL/min，注射完毕后，停针5min，缓慢移除针头。
实施例4 MRI扫描检测
将实施例3表达后的大鼠脑组织进行MRI扫描检测，在病毒注射后5-6周后，对大鼠进行麻醉后进行MRI(uMR790，Shanghai United Imaging Healthcare)扫描，采用立体像素分辨率为0.25*0.25*1.5mm的三维磁化强度预备梯度回波序列，采集大鼠全脑冠状面、水平面的T1加权图像，每个面扫描4-6分钟；结果 见图3(A)-图3(C)，由于本申请GEM表达后能结合锰离子，锰离子为顺磁性物质，因此MRI扫描时采集T1加权像，其中图3(A)为的大鼠脑补冠状面扫描结果，图3(B)为大鼠脑部水平面扫描结果，图3(C)为病毒注射与MRI结果对应图，慢病毒采用表达性较强的泛素启动子Ub，其中Amcyan为荧光蛋白标签，用于检测病毒的表达，图中病毒左侧为对照；结果表明，本申请GEM可以在T1加权像上产生高信号，MRI的信号和病毒表达的信号是重合的。
将本申请与成像效果与原有技术T2加权成像对比检测，结果如图4(A)-图4(B)所示，其中，图4(A)为原有技术T2加权像，图4(B)本申请T1加权成像效果图，由图4(A)-图4(B)可知，与原有技术相比，本申请的分辨率更高，检测结果更直观清晰。
综上所述，本申请提供的小分子蛋白为特殊改造的基因编码的核磁共振成像造影剂，在生物体内表达后，在核磁成像时能造成T1弛像时间的明显缩短，成像时相关部高信号，解决了目前常用核磁共振成像造影剂的组织穿透性差、没有细胞/组织选择性、生物毒性强(金属造影剂)等众多问题，能够高分辨率、实时、在体的检测基因表达、细胞分化个体发育过程等，具有广阔的应用前景和巨大的市场价值。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (12)

  1. 一种小分子蛋白质，氨基酸序列如SEQ ID NO.1所示。
  2. 根据权利要求1所述的小分子蛋白质，其中，所述小分子蛋白质的亲和金属离子包括2价锰离子。
  3. 根据权利要求2所述的小分子蛋白质，其中，所述小分子蛋白质的核苷酸序列如SEQ ID NO.2所示。
  4. 一种质粒，其包括编码权利要求1-3中任一项所述的小分子蛋白质的核苷酸序列。
  5. 一种慢病毒，其通过权利要求4所述的质粒和辅助质粒共转染包装得到。
  6. 如权利要求1-3中任一项所述的小分子蛋白质、权利要求4所述的质粒或权利要求5所述的慢病毒用于制备核磁共振成像造影剂的用途。
  7. 如权利要求1-3中任一项所述的小分子蛋白质、权利要求4所述的质粒或权利要求5所述的慢病毒用于制备神经示踪剂的用途。
  8. 如权利要求1-3中任一项所述的小分子蛋白质、权利要求4所述的质粒或权利要求5所述的慢病毒用于制备检测基因表达的标记物的用途。
  9. 一种如权利要求1-3中任一项所述的小分子蛋白质的制备方法，其包括如下步骤：
    (1)以GEM蛋白的核苷酸序列为基础，进行酶切连接，构建GEM质粒；以及
    (2)将步骤(1)构建的质粒进行蛋白表达。
  10. 根据权利要求9所述的制备方法，其中，步骤(2)所述蛋白表达的方式包括将GEM质粒进行慢病毒包装并感染细胞或注射组织。
  11. 根据权利要求10所述的制备方法，其中，步骤(2)所述蛋白表达的载体包括Hela细胞、大鼠脑组织或恒河猴脑组织中的任意一种或至少两种的组 合。
  12. 一种如权利要求1-3中任一项所述的小分子蛋白质的制备方法，其包括如下步骤：
    (1)以氨基酸序列如SEQ ID NO.1所示GEM蛋白的核苷酸序列为基础，在其3’端和5’端以BamHI和EcoRI两种酶插入pUltra-Smurf载体，进行酶切连接，构建GEM质粒；以及
    (2)将步骤(1)构建的质粒进行慢病毒包装，并进行蛋白表达。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111233982A (zh) * 2018-11-28 2020-06-05 深圳先进技术研究院 一种小分子蛋白质及其应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109402075A (zh) * 2018-11-28 2019-03-01 深圳先进技术研究院 一种用于清除氧自由基的小分子蛋白质及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101203249A (zh) * 2005-04-01 2008-06-18 德克萨斯大学体系董事会 多（肽）作为螯合剂：制造方法和用途
CN101222878A (zh) * 2005-07-13 2008-07-16 佐治亚州立大学研究基金会 显像剂及其制备方法
JP2009100718A (ja) * 2007-10-25 2009-05-14 National Institute Of Advanced Industrial & Technology 蛋白質の製造方法
CN102639152A (zh) * 2009-12-18 2012-08-15 通用电气医疗集团股份有限公司 锰螯合物及其作为磁共振成像(mri)中造影剂的用途
CN107441512A (zh) * 2017-09-22 2017-12-08 合肥工业大学 一种顺磁性一氧化锰纳米颗粒的制备方法及其应用
CN107952084A (zh) * 2017-12-21 2018-04-24 同济大学 一种以人体内源性蛋白为螯合剂的磁共振造影剂及其制备

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102406950A (zh) * 2011-11-29 2012-04-11 中国科学院上海硅酸盐研究所 一种锰基核磁共振成像造影剂及其制备方法和应用
CN111233982B (zh) * 2018-11-28 2023-03-03 深圳先进技术研究院 一种小分子蛋白质及其应用
CN111234028B (zh) * 2018-11-28 2023-03-03 深圳先进技术研究院 一种用于指示钙离子的小分子蛋白质及其应用
CN109402075A (zh) * 2018-11-28 2019-03-01 深圳先进技术研究院 一种用于清除氧自由基的小分子蛋白质及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101203249A (zh) * 2005-04-01 2008-06-18 德克萨斯大学体系董事会 多（肽）作为螯合剂：制造方法和用途
CN101222878A (zh) * 2005-07-13 2008-07-16 佐治亚州立大学研究基金会 显像剂及其制备方法
JP2009100718A (ja) * 2007-10-25 2009-05-14 National Institute Of Advanced Industrial & Technology 蛋白質の製造方法
CN102639152A (zh) * 2009-12-18 2012-08-15 通用电气医疗集团股份有限公司 锰螯合物及其作为磁共振成像(mri)中造影剂的用途
CN107441512A (zh) * 2017-09-22 2017-12-08 合肥工业大学 一种顺磁性一氧化锰纳米颗粒的制备方法及其应用
CN107952084A (zh) * 2017-12-21 2018-04-24 同济大学 一种以人体内源性蛋白为螯合剂的磁共振造影剂及其制备

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"hypothetical protein [Burkholderia thailand ensis", NCBI REFERENCE SEQUENCE: WP-011401419.1, 16 May 2013 (2013-05-16) *
SI , M. ET AL.: "Manganese scavenging and oxidative stress response mediated by type VI secretion system in Burkholderia thailand ensis", PNAS, 27 February 2017 (2017-02-27), pages E2233 - E2242, XP55712522 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111233982A (zh) * 2018-11-28 2020-06-05 深圳先进技术研究院 一种小分子蛋白质及其应用
CN111233982B (zh) * 2018-11-28 2023-03-03 深圳先进技术研究院 一种小分子蛋白质及其应用

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